Membrane Restructuring Research Articles

Phospholipid membrane restructuring induced by saposin C: a topographic study using atomic force microscopy

The enzymatic activity of glucosylceramidase depends on the presence of saposin C (Sap C) and acidic phospholipid-containing membranes. In order to delineate the mechanism underlying Sap C stimulation of the enzyme activity, it is important to understand how Sap C interacts with phospholipid membranes. We studied the dynamic process of Sap C interaction with planar phospholipid membranes, in real time, using atomic force microscopy (AFM). The phospholipid membrane underwent restructuring upon addition of Sap C. The topographic characteristics of the membrane restructuring include the appearance of patch-like new features, initially emerged at the edge of phospholipid membranes and extended laterally with time. Changes in the image contrast of the phospholipid membrane observed after the Sap C addition indicate that a new phase of lipid–protein structure has formed during membrane restructuring. The process of membrane restructuring is dynamic, commencing shortly after Sap C addition, and continuing throughout the duration of AFM imaging (about 30 min, sometimes over 1 h). This study demonstrated the potential of AFM real-time imaging in studying protein–membrane interactions.

Real-Time Observation of Phospholipid Bilayer Membrane Restructuring Induced by Protein Molecules using Atomic Force Microscopy

Atomic force microscopy (AFM) allows the surfaces of native biological materials to be imaged in aqueous solution with submolecular resolution. The ability to perform AFM imaging in aqueous and physiological environment has made it possible to monitor important biological processes in real time at high resolution. Currently, there is a great deal of interest in AFM studies of the structure and property of lipid bilayer membranes and protein interactions with lipid bilayer membranes. Lipid bilayer membranes in biological cells form a permeability barrier, which controls the flow of ions, water, and other molecules between biological cells and their environments, whereas membrane-bound and/or membrane-associated proteins are responsible for most of the dynamic functions carried out by the membrane. However, real-time AFM monitoring of dynamic biological processes has been challenged by the limited temporal resolution of AFM, potential physical damage to soft biological samples, and intrinsic complexity of biological processes. There are few successful examples of AFM real-time studies of dynamic biological events, particularly in the aspect of protein interactions with lipid bilayer membranes.We have attempted to use atomic force microscopy to study interactions between a particular protein, saposin C, and phospholipid bilayer membranes in real time. Saposin C (Sap C), a small glycoprotein, is an essential co-factor for the hydrolysis of glucosylceramide by glucosylceramidase in lysosomes, and a deficiency of Sap C leads to a variant form of Gauchers’ diseases. Supported planar phospholipid bilayer membranes were used in the study.

The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia.

Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the gamma-isoform of actin in unstimulated cells, to the beta-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.

Membrane Lipids: What Membrane Physical Properties are Conserve during Physiochemically-Induced Membrane Restructuring?

SYNOPSIS. Current theories assert that organisms finely adjust the order, or fluidity, of their cellular membranes in response to changes in their physiochemical environment ( e.g. , pressure, temperature, salinity, etc.). However, membrane order may not be the only property that is conserved. The most commonly observed alterations in cell membrane composition under conditions of altered physiochemical environment, namely changes in the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio and the content of highly unsaturated acyl chains, are difficult to fully reconcile with the conservation of membrane order alone. This report reviews the literature concerning two properties of membranes that may play vital roles in the adaptation of cellular membranes to changing environments: a ) the tendency of membranes to relax into the reversed hexagonal phase and b ) the occurrence and structure of lipid-driven domains within the membrane. The tendency of a membrane to form the reversed hexagonal phase is a property central to a variety of important cellular events. This tendency is tightly regulated by variation of the ratio of hexagonal phase-forming lipids to lamellar phase-forming lipids in the membrane. In most animal cells, this corresponds to the PE/PC ratio. Highly unsaturated acyl chains, in conjunction with cholesterol, modulate the occurrence and structure of lipid-driven membrane domains. These membrane domains are also critically involved in a number of key cellular processes. The changes in membrane lipid composition that occur during adaptation to the environment may be required for the preservation of the tendency to form nonlamellar phases and of the occurrence and specific structure of domains within the membrane, in addition to overall membrane order.

MEMBRANE STRUCTURAL LIPID CHANGES IN FRESH-CUT CARROTS: REVISITING THE "WOUNDING AND AGING" PHENOMENON

Virtually no quantitative data exist for membrane lipid metabolism of fresh-cut (wound-stressed) vegetables and fruits, despite the inevitable consequences of wounding. One such result is the rupture of cell membranes and activation of membrane lipid catabolic processes. We have evaluated changes in membrane sterols and glycerolipids in shredded carrots, a popular fresh-cut commodity. 'Apache' and 'Caropak' carrots were shredded and stored under conditions similar to those used in retail markets (10°C and 95% RH). Between 0 and 10 days of storage, membrane lipid concentrations in the shredded tissue were determined using HPLC/evaporative light scattering detection. Total phospholipid and acylated sterol glycoside concentrations increase by as much as 31% and 135%, respectively, following 10 days of storage. These increases are indicative of membrane restructuring (repair) processes, are accentuated by Ca 2+ pretreatment, and appear to be cultivar-dependent. During storage, Ca 2+ pretreatment also delays onset of increases in the ratios of free sterols to phospholipids and of free stigmasterol to free sitosterol (both senescence-driven processes); and as expected, Ca 2+ pretreatment reduces cell permeability. However, Ca 2+ pretreatment does not curb the rate of accumulation of phosphatidic acid, a membrane degradation product. Coexistence of membrane degradation and repair processes appears to be a characteristic feature of wounding and long-term storage of carrots. Further, Ca 2+ pretreatment may prove to be a valuable approach for maintaining quality of fresh-cut carrots and possibly other fresh-cut products, owing to its stabilising influence on cell membrane systems.

Plasticity of the properties of mitochondria from rainbow trout red muscle with seasonal acclimatization

Cold-acclimation of rainbow trout brings only limited changes in muscle metabolic capacities, but marked modifications in membrane composition. Thus, we examined whether the functional properties of mitochondria from trout red muscle were modified by seasonal temperature acclimatization. Mitochondria from fall-acclimatized trout had higher maximal capacities (state 3 rates) for the oxidation of pyruvate and acyl carnitines at 12 and 20 °C than mitochondria isolated from summer-acclimatized trout. For these substrates, the increased oxidative capacity completely compensated for the seasonal drop in temperature. Pyruvate and palmitoyl carnitine were consistently the preferred substrates, while decanoyl and octanoyl carnitine were oxidized at higher rates than glutamine, particularly in fall trout. State 4 rates of oxygen uptake (obtained when all ADP has been converted to ATP) differed less among substrates, but varied seasonally. State 4 rates at 12 and 20 °C were higher in mitochondria isolated from fall than summer trout. At low temperatures, the Q10 of both maximal and state 4 rates of substrate oxidation tended to be higher for mitochondria from fall trout. The apparent Arrhenius activation energy (Ea) for mitochondrial pyruvate oxidation was higher in fall than summer trout whereas the Ea’s for palmitoyl carnitine and decanoyl carnitine oxidation did not change. The fatty acids of mitochondrial phospholipids from fall trout were more polyunsaturated than those from summer trout, with 12% more double bonds occurring than in summer trout, suggesting that membrane restructuring may be involved in the observed compensatory responses.

Calcium delays senescence-related membrane lipid changes and increases net synthesis of membrane lipid components in shredded carrots

The influence of Ca2+ on firmness and membrane lipid structural components was evaluated during storage of shredded (wound-stressed) carrot tissues (Daucus carota L. ‘Caropak’). During 10 days of storage at 10 °C and 95% RH, Ca2+ treatment (applied as 1% w/v CaCl2 upon shredding) provided 6–16% greater firmness retention than did a water control treatment. Calcium accentuated net increases in total phospholipid and monogalactosyldiacylglycerol concentrations after 4 and 10 days of storage, and in acylated steryl glycoside concentrations after 4 days of storage. Calcium partially counteracted a net reduction in steryl glycoside concentration which occurred following 4 days of storage, and prevented an increase in the free sterol : phospholipid ratio following 10 days of storage. Calcium preserved membrane integrity of carrot shreds not only by delaying senescence-related membrane lipid changes, but also by apparently augmenting membrane restructuring processes.

Restructuring of plasma membrane phospholipids in isolated hepatocytes of rainbow trout during brief in vitro cold exposure

Adaptive changes in membrane physical properties in response to changing environmental temperature (e.g., inereased fluidity at low growth temperatures) are well known in poikilotherms; however, the timecourse of this response has received little attention. In this study the plasma membrane lipids of hepatocytes prepared from 20°C-acclimated trout were analyzed for phospholipid class and molecular species composition and metabolism after the cells were exposed to 5°C for 6 hours. Proportions of phosphatidylethanolamine and phosphatidylcholine were not altered by in vitro incubation at either 5 or 20°C. Molecular species analysis revealed that proportions of 18:1/20:5-phosphatidylcholine were significantly lower in plasma membranes of 5°C incubated cells, while decreases in 16:0/20:4-phosphatidylcholine, an unidentified phosphatidylcholine species, and 16:0/16:0-phosphatidylethanolamine as well as increases in 16:0/16:1-phosphatidylethanolamine as well as increases in 16:0/16:1-phosphatidylcholine bordered on significance. Exogenous radiolabeled molecular species of phosphatidylcholine (16:0/16:0-phosphatidylcholine and 16:0/18:1-phosphatidylcholine) were converted into other species at both temperatures, and the formation of some was influenced by incubation temperature. For example, cells exposed to 5°C convert significantly more 16:0/16:0-phosphatidylcholine into 16:0/20:4-phosphatidylcholine and 18:0/16:1-phosphatidylcholine and less into 18:1/18:1-phosphatidylcholine and 16:0/22:6-phosphatidylcholine than cells incubated at 20°C. In addition, cells at 5°C metabolized 16:0/18:1-phosphatidylcholine to a lesser extent than those at 20°C. The profile of conversion products indicates that deacylation/reacylation, elongation and desaturation reactions all participate in this early membrane restructuring. It is concluded that the plasma membrane of trout hepatocytes is a highly dynamic structure characterized by continuous lipid restructuring/turnover which can be rapidly altered upon acute cold exposure to adjust membrane phospholipid molecular species composition to the prevailing thermal environment.

Delta-Haemolysin from Staphylococcus aureus and model membranes. A solid-state 2H-NMR and 31P-NMR study

Solid-state 2H NMR and 31P NMR of 2H-enriched chains and polar head groups, respectively, of dipalmitoylglycerophosphatidylcholine/water dispersions were undertaken to investigate the action of delta-haemolysin from Staphylococcus aureus on biomembranes. When the lipid/toxin molar ratio, Ri, is greater than or equal to 10, the gel-phase 2H powder patterns and the temperature of the gel-fluid phase transition, tc, are unchanged by the presence of the toxin whereas the 31P powder spectra of polar head groups are perturbed. At t greater than tc, a detailed analysis of methylene ordering indicates that delta-haemolysin orders the lipid chains near tc and disorders them for t much greater than tc. These findings are interpreted in terms of peptide location with regard to the membrane and suggest that the position of the toxin depends on the temperature relative to tc. Experiments carried out at Ri = 4 exhibit sharp, isotropic 2H-NMR lines, at t greater than tc, indicating that delta-haemolysin promotes the appearance of very small objects undergoing fast isotropic reorientation which average to zero the quadrupolar interaction. Below tc, one observes gel-phase powder patterns which indicate that the bacterial toxin is unable to form such small structures with ordered dipalmitoylglycerophosphocholine phospholipids. From comparison of the action of delta-haemolysin with that of melittin on same lipids [Dufourc et al. (1986) Biochemistry 25, 6448-6455] it results that both toxins perturb similarly fluid-phase lipids at elevated temperature, but they behave differently with gel-phase lipids, the former toxin being less efficient in membrane restructuring than the latter.

Adaptations to temperature in the cellular membranes of crustacea: membrane structure and metabolism

1. 1.|Acute changes in environmental temperature have deleterious effects on the structure and function of cellular membranes of crustaceans, as well as other poikilothermic animals. Low temperature increases membrane order, and decreases “fluidity”, in addition to influencing the activity of membrane-bound enzymes. 2. 2.|Crustaceans adapt to prolonged exposure to low temperatures by increasing the fluidity of membrane lipids, which serves to restore membrane function. 3. 3.|Membrane fluidity is regulated by changing the proportions of saturated and unsaturated fatty acids on membrane phosphatides at different temperatures. Unsaturation increases at low temperatures. 4. 4.|The proportion of different phospholipid head groups also changes in response to temperature, particularly among winter-active crustaceans. Ethanolamine phosphatides increase at the expense of choline phosphatides at low temperatures. 5. 5.|The experimental evidence for various metabolic mechanisms of membrane restructuring in crustaceans is discussed.

The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: Analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.